The genome sequence of the springtail, Dicyrtomina minuta (O.Fabricius, 1783)

We present a genome assembly from an individual female Dicyrtomina minuta (springtail; Arthropoda; Collembola; Symphypleona; Dicyrtomidae). The genome sequence is 582.0 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X 1 and X 2 sex chromosomes. The mitochondrial genome has also been assembled and is 15.59 kilobases in length.


Background
Springtails are one of the most abundant groups of soil animals, found in all sorts of biomes and habitats worldwide (Hopkin, 1997).Most of the springtails are truly miniature creatures, often smaller than a millimetre, but there are exceptions.Dicyrtomina minuta is one of the larger globular springtails and is up to 3mm long, which is a paradox given its species name (since minuta means small).These springtails can be found in moist leaf litter in Europe and North America during autumn and winter, alongside other species of the same family Dicyrtomidae (GBIF Secretariat, 2024).The fourth antenatal segment of Dicyrtomidae is very short, which is a distinct feature that can be used for quick identification to family level.Another remarkable trait of the family is the secretion of numerous rods of wax, secreted by special chaetae (Massoud & Vannier, 1965).The rods are sheddable, and their function remains unclear, presumably involved in the sensorial system.Three species of the Dicyrtomina genus, D. minuta, D. ornata and D. saudersi differ only in colour pattern variations and therefore were considered by some authors morphs of the same species (Richards, 1968).
In general, karyotypes of globular springtails (Symphypleona) are thought to be conserved, with 2n=12 chromosomes in females and 2n=10 in males (Dallai et al., 2004), which was also observed in D. ornate (Dallai et al., 1999).The only exception is a more distantly related member Dicyrtomidae, Ptenothrix italica, with 2n=14 and 2n=12 in males (Dallai et al., 1999).However, the karyotype of D. minuta was prior to this study unknown.Based on the overall conservation of the double X chromosome system (X 1 X 2 00) and aberrant spermatogenesis in Symphypleona (Dallai et al., 1999), it is expected that this species is reproducing via Paternal Genome Elimination (Jaron et al., 2022), a reproductive mode where males eliminate the paternal genome, and pass only the maternal one to the next generation.However, this hypothesis has not been directly tested in this species yet.
The genome of a springtail, Dicyrtomina minuta, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Dicyrtomina minuta, based on a female specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from an adult female Dicyrtomina minuta collected from Wytham Woods, Berkshire, UK (51.78, -1.34).A total of 39-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 68 missing joins or mis-joins and removed 8 haplotypic duplications, reducing the assembly length by 0.70% and the scaffold number by 6.36%, and increasing the scaffold N50 by 2.24%.
The final assembly has a total length of 582.0 Mb in 485 sequence scaffolds with a scaffold N50 of 89.8 Mb (Table 1).The snail plot in Figure 1 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 2. The cumulative assembly plot in Figure 3 shows curves for subsets of scaffolds assigned to different phyla.Most (88.88%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing three autosomes and the X 1 and X 2 sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 4; Table 2).Chromosomes X 1 and X 2 were identified based on synteny with Allacma fusca (GCA_947179485.1)(Jaron et al., 2023).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
Specimens of Dicyrtomina minuta were collected from Wytham Woods, Oxfordshire, UK (latitude 51.78, longitude -1.34) on 2020-08-03 by aspiration.The specimens were collected and identified by Kamil Jaron (Wellcome Sanger Institute) and preserved on dry ice.The specimen used for genome sequencing was a female adult Dicyrtomina minuta (specimen ID Ox000721, ToLID qeDicMinu4), and a second specimen (ID Ox000728, ToLID qeDicMinu3) was used for Hi-C sequencing.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the qeDicMinu4 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA  High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument.Hi-C data were also generated from whole organism tissue of qeDicMinu3 using the Arima v2 kit.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.

Genome assembly and curation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups, without the -e option (Guan et al., 2020).The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any  potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

José M Martín-Durán
Queen Mary University of London, London, UK This Genome Note reports the sequencing and assembly of the genome of the springtail (Collembola) Dicyrtomina minuta.Collembolans are fascinating animals that occupy a key phylogenetic position to investigate the evolution of Insecta, the most diverse animal clade on Earth.

Strengths
-High-quality genome for an important and understudied animal group.
-Important resource to investigate sex determination and karyotype evolution in Collembola.
Weaknesses -None for this resource.The fact that the karyotype inferred from genome sequence differs so dramatically from those observed in other species warrants further investigations and experimental validations.
Is the rationale for creating the dataset(s) clearly described?

Alex Makunin
Wellcome Sanger Institute, Hinxton, England, UK The data note by Jaron et al. presents a high quality chromosome scale assembly for Collembola species Dicyrtomina minuta.This is the first chromosomal genome assembly for Dicyrtomidae family.The presentation of the results and methods is standardized and overall very transparent.

I have a few minor suggestions:
Is it possible to specify the amount of DNA used for PacBio sequencing?From the methods section it seems that the no amplification was done prior to sequencing, i.e. low input protocol was used.

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The identification of X1 and X2 chromosomes is based on genome alignment to Allacma fusca, also from DToL project (Jaron et al., 2023).However, in that publication the source of chromosome identities is not specified, so it seems reasonable to include more information on sex chromosomes delineation -I assume it was based on coverage given that a male Allacma fusca was used for assembly.

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It might be worth including a synteny plot as a figure, possibly accompanied by information on expected divergence times between Dicyrtomina (Dicyrtomidae) and Allacma (Sminthuridae), e.g.estimate of ca 280MY from mitochondrial genomes (Leo et al., 2019).

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It might be interesting to include a short discussion around expected chromosome numbers and their evolutionary variability.For example, D. ornata females were reported to have 2n = 12 (Dallai et al 1999) -one chromosome more than reported for D. minuta here, although in introduction it is mentioned that both were considered as a single species at some point Reviewer Expertise: Comparative genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Genome assembly of Dicyrtomina minuta, qeDicMinu4.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 581,995,013 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (181,988,040 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (89,754,956 and 1,302,000 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the arthropoda_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Dicyrtomina%20minuta/dataset/qeDicMinu4_1/snail.

Figure 2 .
Figure 2. Genome assembly of Dicyrtomina minuta, qeDicMinu4.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Dicyrtomina%20minuta/dataset/qeDicMinu4_1/blob.

Figure 3 .
Figure 3. Genome assembly of Dicyrtomina minuta qeDicMinu4.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Dicyrtomina%20minuta/dataset/qeDicMinu4_1/cumulative.
note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Figure 4 .
Figure 4. Genome assembly of Dicyrtomina minuta qeDicMinu4.1:Hi-C contact map of the qeDicMinu4.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=cRUc7wPZQ9e5NHpn5R-dYg.
KS, Berg MP, Ellers J, Hodson CN, et al.: The genome sequence of the springtail Allacma fusca (Linnaeus, 1758).Wellcome Open Res.2023; 8: 319 PubMed Abstract | Publisher Full Text Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Partly Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Table 1 :
Proposed standards and metrics for defining genome assembly quality" from Rhie et al. (2021).
was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30(Todorovic  et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation(Strickland et al., 2023): in brief, the method employs AMPure PB beads to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.